This invention relates to a method and a kit for the detection and collection of microorganisms from the air and from surfaces utilizing a collection device that employs a dry collection methodology and a dry growth medium, and a container of a premeasured volume of liquid for hydrating the dry growth medium after microorganism collection on the dry growth medium has occurred.
Many molds are considered to be harmful microorganisms. Mold spores are minute propagating units that facilitate the growth and spread of mold colonies. Inhalation of mold spores and other microorganisms into the lungs of humans has been found to be a significant factor in causing a number of different pneumoconioses and other health problems such as allergies, headaches, and fatigue. Most molds require oxygen and water to live and the aerobic molds of our environment are known to multiply and migrate by producing and releasing millions of mold spores into the air.
Methods for estimating the relative environmental mold spore bioburden count exist, but these methods involve the collection of environmental air mold spores on wet media such as Sabouraud""s Dextrose Agar (an aqueous suspension of gelatin and nutrients that solidifies to a semi-solid, water-based gel below 45xc2x0 C.). Exposing sterile Sabouraud Dextrose Agar in an open container such as a petri dish to air containing mold spores can result in mold spores settling on the agar on which they can then germinate and grow. After a number of days, the colonial growth resulting from the germination of a single spore may be so large that the colony may be visually discernable without the use of magnification equipment.
An example of the device described above is disclosed in U.S. Pat. No. 3,968,012 which provides a device for detecting bacteria and other microorganisms carried in the air inside of hospital respiratory machines, such as ventilators and anesthesia gas machines. The device comprises a culture medium dish, a cylindrical casing, and a sanitary cover where the dish and cover are substantially similar to a petri dish. In operation, the device is attached to the vent of a respiratory machine, and the cylindrical casing controls the flow of the air to the medium dish so that microbial particles are collected from the air.
Many other devices in the prior art combine machinery with wet culture media to sample air for testing. For example, U.S. Pat. No. 3,956,070 discloses a device comprising a casing to direct a flow of air over a cartridge containing a culture medium. The cartridge is comprised of a two-sided strip that is wound around two reels with a culture medium spread over each side of the strip. In operation, air is vented through the casing and over both sides of the culture medium strip. U.S. Pat. No. 3,980,524 discloses a device resembling the shape of a common flashlight which comprises a casing that houses batteries, a motor with a drive-shaft and flanges, and a culture medium cup. In this device, the motor is employed with the drive-shaft and flanges to direct a volume of air over the medium cup.
Dry growth media has been used to detect microorganisms in liquid samples. U.S. Pat. No. 4,565,783 discloses a device for growing microorganisms, comprising a self-supporting water-proof substrate, a layer of adhesive coated thereon, and a coating of dry, cold-water-soluble growth medium powder adhered uniformly to the surface of the adhesive. In accordance with the teachings of U.S. Pat. No. 4,565,783, when an aqueous test sample is placed on the substrate in contact with the dry growth medium powder, the growth medium is hydrated to a gel and germination of microorganisms present in the test sample may result.
Since environmental mold spores are and have been associated with adverse health effects in humans, it is desirable to monitor the environmental mold spore bioburden count in dwellings, office buildings, schools and other indoor areas inhabited by people. Various health organizations routinely monitor environmental air in specific locations for mold spores using passive and active agar impaction methods, however, these organizations generally do not test individual homes, businesses, churches, etc. There are health professionals who, for a fee, monitor the air for environmental mold spores in an individuals most frequently inhabited environments, but fees are high and anonymity may not be maintained during and/or after the testing.
Establishing the environmental mold spore count by moist agar impaction methods utilizing Sabouraud Dextrose Agar or other known nutrient formulations, for example trypticase soy agar as disclosed in U.S. Pat. No. 3,968,012, has many associated problems. The first problem is that sealed agar plates utilized in moist agar impaction methods have a relatively short shelf life, on the order of a few weeks at most. Further, moist agar can lose its moisture rapidly over time and, as a result, its ability to support microorganism germination and growth decreases rapidly to a point where germination may not occur at all.
In addition, the use of moist agar to collect environmental mold spores may create what can be described as a time lag problem. An environmental mold spore impacting an unsealed hydrated microbiological growth medium during the first moments of exposure, i.e., the first minute in a 60-minute exposure, may enjoy distinct survival advantages as compared to an environmental mold spore impacting a desiccated microbiological growth medium during the last moments of the exposure as a direct result of changes in moisture conditions of the growth medium over the exposure interval. The latter arriving spores may geminate only very slowly producing a micro-colony that may be too small to be visually enumerated, or may not even germinate at all. An undercount of the actual population of spores can result producing an inaccurate assessment of the true population of environmental mold spores in the location under analysis.
The known methods of the prior art typically employed in air quality sampling are the use of passive agars in petri dishes or agars which are inserted into active volumetric pump samplers. The use of agars in air quality sampling has multiple impediments for the individual consumer/user, including increased costs for preparation and storage and shipping, a very short shelf life and, if active volumetric pump samplers are used, an extremely high purchase cost or rental cost.
A useful method of environmental air and surface sampling which comprises the dry collection and subsequent hydration, growth and enumeration of microorganisms has been discovered. The use of such procedures has been found to overcome the problems of time-lag growth disparities and evaporation of the activating liquid during collection identified above. In addition, dry collection devices exhibit a relatively long shelf-life and provide the added advantage of being easier to handle, especially at the extremes of normal ambient temperatures. The dry collection method of the present invention is accomplished by means of an easy to use, low cost home test kit to quantify the environmental mold spore and microorganism count and represents an important step in the reduction of environmental microorganism levels. Such a kit is also useful to educators teaching concepts of microbiology and mathematics. The method and kit is also a tool for use in monitoring work environments including commercial/industrial facilities.
One aspect of the present invention is a method of detecting microorganisms comprising the steps of exposing a dry collection device containing a dry growth medium to environmental microorganisms, adding a premeasured volume of liquid to the dry growth medium, and allowing collected microorganisms to grow into colonies. In certain embodiments, the exposure step may be accomplished by placing the collection device on a surface for a predetermined interval of time so that microorganisms in the air can settle out onto the collection device. Alternatively, the exposure step may also be accomplished by the direct application of a dry adhesive collection device to a surface so that microorganisms located on the surface can be captured and transferred directly to the dry growth medium prior to hydration. While the step of adding a premeasured volume of liquid to the dry growth medium can be accomplished in a variety of ways all of which are considered to be within the scope of the invention, it is preferred that the liquid is applied so that only a predetermined area of the dry growth medium is hydrated. In such a fashion, the subsequent counting of microorganism colonies can be accomplished with respect to a standardized counting area and populations can be expressed in terms of colonies per unit area. One preferred technique for hydrating the dry growth medium involves the use of a hand press in which the press is placed on the cover over the liquid after its application to the dry growth medium and pressure is applied thereto sufficient to spread the liquid out over a predetermined area of the dry growth medium.
The growth step of the method of the present invention is accomplished by allowing a sufficient interval of time to elapse to permit the growth of microorganism colonies and, depending on the growth characteristics of particular microorganisms which are being detected, can involve the placement of the hydrated collection device in an ambient environment or in a non-ambient environment such as, for example, an incubator. Certain embodiments of the method of the invention further comprise the step of counting the colonies of microorganisms that have grown on the growth medium device after its exposure to air and after hydration of the dry growth medium. The results from the count can then be analyzed in accordance with a variety of parameters known in the art.
Another aspect of the present invention is a kit comprising a microorganism collection device having a substrate and a layer of dry growth medium applied thereon, and a container of a premeasured volume of liquid for hydrating the dry growth medium once collection of microorganisms has occurred. In certain embodiments, the substrate of the device is waterproof and self-supporting, while in other embodiments the substrate is in the form of a tape comprising a non-porous layer and a micro-porous layer adhesively attached thereto. The dry growth medium applied thereon is preferably soluble in cold or ambient water and in the form of a powder. The powder may comprise a gelling agent in sufficient amount to provide a gel having a Brookfield viscosity of at least 1500 cps when hydrated with a premeasured amount of water. The dry growth medium may have sufficient inherent adhesive characteristics such that it may be adhered directly to the substrate. Alternatively, the bond between the dry growth medium and the substrate may be facilitated by means of an adhesive pre-applied to the substrate or by mixing the dry growth medium with an adhesive prior to application to the substrate. In another embodiment, the collection device further comprises an air-permeable membrane. The membrane may improve the growth of certain aerobic microorganisms if the growth medium is covered after exposure and hydration.
In certain embodiments, the collection device may further comprise a cover sheet releasably adhered to at least a portion of the substrate. The cover sheet is opened in the first step of the method to expose the dry growth medium to the environment, and then the cover sheet is closed after the liquid is added to the medium and before the liquid is spread over the medium. The cover sheet serves several functions including the protection of the dry growth medium from unintended premature exposures, the retention of microorganisms once the collection step is completed, the retention of moisture once the hydrating liquid has been applied to the dry growth medium, and the preservation of the culture upon completion of microorganism colony growth.
In certain embodiments, the kit further comprises a hand press for applying the premeasured volume of liquid to a predetermined area of the dry growth medium. While the press may assume a variety of configurations that will accomplish the distribution of the liquid over the dry growth medium, the press preferably will comprise a pressing surface and a raised ring on the pressing surface defining a predetermined area such that the application of the press to the liquid on the growth medium results in the spreading out of the liquid to the full extent of the predetermined area. The resulting area in which the growth medium is hydrated allows for standardized counting so that microorganism populations can be expressed in terms of colonies per unit area. The kit of the invention can assume various configurations based on the needs and desires of the user as the kits may be tailored specifically for home, commercial/industrial, or office use and have applicability as well as teaching aids in schools and particularly for science instruction, all of which are considered to be within the scope of the invention.
The kit of the device is particularly intended for use by consumers without need for assistance or employment of a microbiologist, laboratory technician or other skilled personnel The implementation of the quantifiable collection of environmental microorganisms from air and surfaces is made much more cost effective by employment of the collection methodology of the invention.